RSPO3 overexpression impairs erythropoiesis. Representative FACS plots analyzing erythroid progenitors in bone marrow (A) and spleens (B) from control and RSPO3 mice; n = 20 per group. (C) Timeline of anemia development in RSPO3 animals measured as the percentage of CD71^– Ter119⁺ erythroid cells among mononuclear cells; n = 3 to 5 per time point. (D) Erythropoietin concentration in serum from control and RSPO3 animals; n = 6 per group. (E) Bar graph depicting the number of burst-forming unit–erythroid (BFU-E), colony-forming unit–granulocyte macrophages (CFU-GM), and colony-forming unit–granulocyte erythrocyte monocyte/macrophage megakaryocyte (CFU-GEMM) after culturing equal numbers of total bone marrow cells from control and RSPO3 mice for 10 days in methylcellulose; n = 6 per group. (F) Quantification of CD71⁺ Ter119^– pro-erythroblasts in control and RSPO3 mice; n = 9 per group. (G) Representative FACS plots and corresponding bar graph analyzing various stages of terminal erythropoiesis in control and RSPO3 mice; n = 9 per group. Gates from right to lower left represent basophilic (baso-), polychromatic (poly-), and orthochromatic (ortho-) erythroblasts, followed by reticulocytes (Ret) and RBCs. (H) Absolute number of reticulocytes in peripheral blood from control and RSPO3 mice; n = 14 per group. (I) Bar graph depicting the relative number of erythroid-macrophage islands in the bone marrow of control and RSPO3 animals measured as the percent CD11b⁺FSC^high among Ter119⁺ cells by flow cytometry; n = 3 to 4 per group. Tissues were analyzed 1 month after tamoxifen induction, and data are represented as mean ± standard error of the mean. One-way ordinary analysis of variance for panel C, Mann-Whitney test for panel D, and two-tailed unpaired t test for all other analyses. Ct, control; FITC, fluorescein isothiocyanate; FSC, forward scatter; n.s., not significant (P > .05); PE, phycoerythrin; RSPO3, tamoxifen-induced animals.